Analytical assay methods, including all biochemical assays and all immunoassays, may be classified into two broad assay techniques: homogenous assays in which the reactants are combined as a reaction mixture and the process of identifying the respective end products is made without any separation of chemical components; and heterogenous assays which combine the reactants as a reaction mixture but which require a separation of the reaction products prior to their identification. Between these two broad classes, heterogenous assays have become much more commonly used for analytical research purposes and in clinical/diagnostic applications.
A particularly well known development in heterogenous assays are immunoassays which utilize the ability of specific polyclonal and monoclonal antibodies to combine selectively with antigens and haptenes. The use of such immunological components alone or in combination with other chemical compounds for qualitative and quantitative detection is now firmly established. The classical, and perhaps best known, example of a heterogenous immunoassay method is the radioimmunoassay (hereinafter "RIA") which offers established procedures for detection of a large variety of different proteins, polypeptides, hormones, drugs, and other chemical compositions. All RIA methods, regardless of individual variation, are based on the technique originally described by R. S. Yallow and S. A. Berson [J. Clin. Invest. 39:1157 (1960)]. Although a huge number of different variations using the basic protocol have been developed, the essence of the method relies upon a competition between an analyte of interest and its radionuclide-labeled analogue for a limited number of specific binding sites on an antibody. Under these test conditions, the concentration of the radionuclide-labeled analogue will vary inversely with the concentration of the analyte of interest in the test sample in their respective ability to bind to the specific antibody. Many variations of the competitive binding assay technique including equilibrium techniques, displacement analysis, and sequential saturation have been developed to meet a variety of different applications; a complete description of each of these different procedures is described in Clinical Radioassay Procedures: A Compendium (Paige K. Besch, Editor) The American Association of Clinical Chemists, Inc., 1975.
Since the essential nature of the RIA is a heterogenous assay method, it is required that a separation be made of the reaction products in order to obtain meaningful results. Accordingly, a number of different means for separating the individual chemical reaction products have been devised. In some instances, one of the reactants has been chemically bound to large, solid carriers such as red blood cells and the like which may be suspended in the reaction fluid and may be radiolabeled; in other techniques, the radiolabeled analogue or a radiolabeled antibody is immobilized onto the surface of a solid substrate. As a result, there has been a long history of developments for immobilization of a reactant directly or via the use of "linker-arms" to solids such as plastic test tubes and disks, agarose and plastic beads, porous glass, and polyacrylamide gels. [Methods In Enzymology, Academic Press, 1980; Updike, Antibiotics and Chemotherapeutics 26:67 (1979); U.S. Pat. Nos. 3,793,445; 3,970,429; and 4,138,474].
Subsequent to the development of the RIA, a wide variety of "labels" other than radionuclides, have been introduced into heterogenous assay systems for a variety of purposes. Common examples of these non-isotopic labels include fluorescent dyes, electron spin radicals, specific binding protein pairs (such as avidin and biotin), and an entire host of different enzymes used in conjunction with their respective co-factors and specific substrates. The last of these has unquestionably become the most well-known in the immunoassay art as the enzyme-linked immuno-sorbent assay or "ELISA".
Despite all these improvements in heterogenous assays, each of the presently known and used assay methodologies places undesirable restrictions and limitations on the user. For example, only the RIA technique provides an analytical sensitivity in the picogram (10.sup.-12 gram) level while the other labeled assay techniques provide reproducible results only in the microgram (10.sup.-6 gram) range. Unfortunately, the radionuclide label must be carefully handled and the problem of waste disposal has risen to an almost insurmountable degree. All of the presently used methods employing one of the known labels requires the use of well trained, skilled technicians and sophisticated detection equipment for accurate results. Moreover, none of the presently known methodologies are particularly useful in the field and cannot be employed on-site for either qualitative or quantitative determinations. It is apparent therefore, that in heterogenous assay methods generally and with immunoassays in particular, there remains a well recognized and continuing need for novel "labels" and new methods for detection and quantitation of an analyte.